以核酸序列分析技術檢測幽門螺旋桿菌抗藥性

Detection of Drug Resistance in Helicobacter pylori by DNA-based Approaches

幽門螺旋桿菌可引起各種胃腸道疾病。目前治療此菌的方法主為三合一療法，包括質子幫浦阻斷劑（proton pump inhibitors, PPI）再合併兩種抗生素。其中治療的第一線藥物為PPI，clarithromycin及amoxicillin，而第二線為PPI，levofloxacin及amoxicillin。因應抗生素施用所產生的細菌抗藥性是造成治療失敗的主因，因此提供藥敏試驗的結果對選擇性用藥將有助益。此菌因生長緩慢，培養及後續藥敏試驗結果非常耗時，現有分生技術的發展，可藉由偵測抗藥基因的熱突變位點來預知抗生素感受性。欲瞭解分生技術施用於本院之可行性，本研究針對clarithromycin及levofloxacin之抗藥基因23S rRNA及DNA gyraseA，利用Real-time PCR及DNA定序法分別加以偵測。研究收集106個胃部黏膜組織塊，直接分析抗藥基因的突變類型，再由此組織塊培養出幽門螺旋桿菌菌株進行藥敏試驗E-test。於106個檢體中有55個培養成功，故培養的陽性率為52%，後續藥敏結果顯示在clarithromycin藥敏試驗中7個敏感性（Susceptible）菌株其23S rRNA基因型皆為野生型；48個抗藥性（Resistant）者其基因型為突變型，clarithromycin表現型和23S rRNA基因型相關性為100%。另levofloxacin 22個表現型為Susceptible者，其DNA gyraseA在87及91號胺基酸上皆無變異；33個表現型為Resistant者有32個在第87或91號胺基酸上皆變異，其中有20個在87號上，有10個在91號，及有2個兼具87和91號變異；而1個Resistant者在87或91號胺基酸上並無變異。Levofloxacin表現型和DNA gyraseA基因型相關性為98.2%。結果顯示基因型和表現型在此兩個抗藥基因上有很高的相關性，另培養失敗的51個案皆可直接分析抗藥基因的類型，因此可由胃部黏膜組織塊直接偵測抗藥突變基因，加快臨床醫師選擇合適藥物做後續治療。 Helicobacter pylori is a Gram-negative, helical shape bacterium infecting the stomach. Standard therapy combines a proton pump inhibitor and two antibiotics, where first-line therapy is consisting of proton pump inhibitors and the antibiotics clarithromycin and amoxicillin (or metronidazole), and the second line antibiotics Fluoroquinolones, such as levofloxacin is often used for rescue therapy. Antibiotic resistance is the major cause of treatment failure for H. pylori. Susceptibility test results helped the right medication; however, H. pylori is a fastidious microorganism and susceptibility results performed after culture is time consuming. Numerous molecular-based techniques have been developed to detect these mutations. Clarithromycin resistance in H. pylori is due to point mutations in the A2143G, A2142G, A2142C of 23S rRNA and levofloxacin resistance in H. pylori is due to point mutations in the DNA gyraseA gene, mainly at codons 87 and 91. We evaluated the efficacy of real-time PCR/melting curve analysis on detection of the most common point mutations in the 23S rRNA for resistance to clarithromycin and DNA sequencing in the DNA gyraseA for resistance to levofloxacin. A total of 106 gastric biopsy specimens were collected in this study. The culture method was successful in 52% (55/106) of H. pylori. 7 clarithromycin susceptible strains were wild type in 23S rRNA gene, 48 resistant strains had mutant genotype. The correlation between 23S rRNA genotyping and clarithromycin susceptibility test was 100%. For levofloxacin, 22 susceptible strains showed no variation at codon 87 and 91 of DNA gyrase A. In addition, variations at codon 87 or 91 were detected in 97.0% (32/33) levofloxacin-resistant strains. Concisely, the consistency of DNA gyraseA genotyping and the levofloxacin susceptibility test can up to 98.2%. 20 strains with mutation at codon 87, 10 strains with mutation at codon 91, and 2 strains mutated at both codon 87 and 91. The results of genotyping in 23S rRNA and the DNA gyraseA had highly correlation with susceptibility test of clarithromycin and levofloxacin. Direct genotyping in these genes of gastric biopsy specimens provides rapid and precise results for the reference of clinical treatment.